Phenolphthalein and bisacodyl: assessment of genotoxic and carcinogenic responses in heterozygous p53 (+/-) mice and syrian hamster embryo (SHE) assay.

Phenolphthalein (800 and 2400 mg/kg/day by gavage and 2400 mg/kg/day by diet) and bisacodyl (800-500, 4000-2000, and 8000 mg/kg/day by gavage) were administered to 15 male and 15 female and 20 male and 20 female p53(+/-) mice respectively for 26 weeks to investigate the potential carcinogenicity of each compound. Toxicokinetic analyses confirmed systemic exposure. p-Cresidine was administered by gavage (400 mg/kg/day) and served as the positive control agent in each study. Dietary phenolphthalein reduced survival in both sexes and early deaths were attributed to thymic lymphoma. No bisacodyl-related neoplasms were observed. Regardless of route of administration to p53(+/-) mice, phenolphthalein but not bisacodyl was unequivocally genotoxic, causing increased micronuclei in polychromatic erythrocytes. In the Syrian hamster embryo (SHE) cell transformation assay, phenolphthalein caused increases in morphologically transformed colonies, thereby corroborating NTP's earlier reports, showing phenolophthalein has potential carcinogenic activity. Bisacodyl was negative in the SHE assay. Results of these experiments confirm an earlier demonstration that dietary phenolphthalein causes thymic lymphoma in p53(+/-) mice and show that (1) phenolphthalein causes qualitatively identical results in this transgenic model regardless of route of oral administration, (2) phenolphthalein shows evidence of micronucleus induction in p53(+/-) mice for up to 26 weeks, (3) phenolphthalein induced transformations in the in vitro SHE assay, and (4) bisacodyl in p53(+/-) mice induces neither drug-related neoplasm, nor micronuclei in polychromatic erythrocytes, and did not induce transformations in the in vitro SHE assay.

Flow cytometric enumeration of micronucleated reticulocytes: high transferability among 14 laboratories.

This laboratory previously described a single-laser flow cytometric method, which effectively resolves micronucleated erythrocyte populations in rodent peripheral blood samples. Even so, the rarity and variable size of micronuclei make it difficult to configure instrument settings consistently and define analysis regions rationally to enumerate the cell populations of interest. Murine erythrocytes from animals infected with the malaria parasite Plasmodium berghei contain a high prevalence of erythrocytes with a uniform DNA content. This biological model for micronucleated erythrocytes offers a means by which the micronucleus analysis regions can be rationally defined, and a means for controlling interexperimental variation. The experiments described herein were performed to extend these studies by testing whether malaria-infected erythrocytes could also be used to enhance the transferability of the method, as well as control intra- and interlaboratory variation. For these studies, blood samples from mice infected with malaria, or treated with vehicle or the clastogen methyl methanesulfonate, were fixed and shipped to collaborating laboratories for analysis. After configuring instrumentation parameters and guiding the position of analysis regions with the malaria-infected blood samples, micronucleated reticulocyte frequencies were measured (20,000 reticulocytes per sample). To evaluate both intra- and interlaboratory variation, five replicates were analyzed per day, and these analyses were repeated on up to five separate days. The data of 14 laboratories presented herein indicate that transferability of this flow cytometric technique is high when instrumentation is guided by the biological standard Plasmodium berghei.

Oxymetholone: I. Evaluation in a comprehensive battery of genetic toxicology and in vitro transformation assays.

Oxymetholone is generally assumed to be a nongenotoxic carcinogen. This assumption is based primarily on the results of an Ames test, existing data in repeat-dose toxicology studies, and the predicted results of a 2-yr National Toxicology Program (NTP) rat carcinogenicity bioassay. To provide a comprehensive assessment of its genotoxicity in a standard battery of mutagenicity assays, oxymetholone was tested in microbial and mammalian cell gene mutation assays, in an in vitro cytogenetics assay (human lymphocytes), and in an in vivo micronucleus assay. Oxymetholone was also tested in an in vitro morphologic transformation model using Syrian hamster embryo (SHE) cells. These studies were initiated and completed prior to the disclosure of the results of the NTP bioassay. Oxymetholone was tested at doses up to 5,000 microg/plate in the bacterial plate incorporation assay using 4 Salmonella strains and the WP2 uvrA (pKM101) strain of Escherichia coil. There was no induction of revertants up to the highest dose levels, which were insoluble as well as toxic. In the L5178Y tk+/- mouse lymphoma assay, doses up to 30 microg/ml reduced relative survival to approximately 30% with no increase in mutants. Male or female human lymphocytes were exposed in vitro to oxymetholone for 24 hr without S9 or 3 hr with S9 and evaluated for the induction of chromosomal aberrations. There was no increase in aberration frequency over control levels and no difference between male and female cells. Peripheral blood from Tg.AC transgenic mice treated dermally for 20 wk with 0, 1.2, 6.0, or 12.0 mg/day of oxymetholone and from p53 transgenic mice treated orally by gavage for 26 wk with 125, 625, or 1,250 mg/kg/day of oxymetholone was evaluated for micronuclei in polychromatic and normochromatic erythrocytes. There was no difference in micronuclei frequency between control and treated animals. These results confirm that oxymetholone is not genotoxic in a comprehensive battery of mutagenicity assays. In the SHE assay, oxymetholone produced a significant increase in morphologically transformed colonies at dose levels of 13-18 microg/ml. The lack of genotoxicity of oxymetholone, the positive response in the in vitro transformation assay, and the results of transgenic mouse carcinogenicity assays will provide an interesting perspective on the results of an on-going NTP rat carcinogenicity bioassay.

Selection of agar for use in Salmonella typhimurium and Escherichia coli mutation assays.

The spontaneous and induced revertant frequency of four Salmonella typhimurium strains (TA1535, TA1537, TA98, and TA100) and Escherichia coli [WP2 uvrA (pKM101)] was evaluated using Vogel Bonner minimal plates prepared with ten different agars. In addition to the Difco Bacto agar originally recommended by Ames, Difco Noble, granulated and Bitek agars; BD grade A, BBL granulated and purified agars; Oxoid purified and No. 1 agars; and GIBCO select agar were tested. Several of these agars have been reported as acceptable alternatives for these Salmonella strains, but comparable studies with E. coli have not been done. The bacteria were treated with DMSO or an appropriate positive control in the presence or absence of an Aroclor 1254-induced rat liver activation system. With the exception of Noble agar in the presence of S9, there was little difference among the responses of the Salmonella strains on any of the agars. However, with E. coli the responses include either a reduction or an increase in spontaneous revertants numbers as well as a reduction in absolute and relative induced revertant frequency. Difco Bacto agar appears to be the most consistent agar for use with these strains. As an alternative, only BBL purified agar resulted in consistent results for all of these strains under all testing conditions. These results emphasize the need to evaluate the components of the standard mutation assay when incorporating additional bacterial strains. Suboptimal responses related to the agar or other components could compromise the detection of weak mutagens.

A direct comparison of mouse and rat bone marrow and blood as target tissues in the micronucleus assay.

Rats and mice were treated concurrently with mitomycin C at a dose of 1 mg/kg/day i.p. for 3 days, a regimen known to induce micronuclei in polychromatic erythrocytes (MN-PCE) in the bone marrow of rats and mice and the peripheral blood of mice. The incidence of micronuclei was evaluated in the peripheral blood and the bone marrow of both species. Early reports suggested that the efficiency of the rat spleen in removing micronuclei from the circulation precluded the use of rat peripheral blood in the detection of chemically-induced micronuclei. The data in the present study demonstrate that the induction of micronuclei in polychromatic erythrocytes as the result of treatment with a clastogen can be demonstrated equally well in the bone marrow or the peripheral blood of both rats and mice.

Genotoxic effects of p-aminophenol in Chinese hamster ovary and mouse lymphoma cells: results of a multiple endpoint test.

p-Aminophenol (PAP), a metabolite of aniline and acetaminophen, has been reported to be mutagenic in the L5178Y mouse lymphoma assay, but not in the CHO/HGPRT assay. In the present study, the effects of PAP in these two cells lines were examined to determine if the difference in activity is related to an intrinsic difference in the cell lines. CHO and L5178Y tk +/- mouse lymphoma cells were treated with PAP for 4 hr and assayed for 3 genetic endpoints: gene mutation at the HGPRT or TK locus, respectively; chromosomal aberrations at approximately 20 hr after initiation of treatment; and single-strand DNA breaks as detected by the single cell electrophoresis assay immediately after treatment. All treatments were conducted in the absence of S9. There was a dose-related, significant increase in TFT-resistant mouse lymphoma cells at dose levels that reduced survival to < or = 50% of concurrent controls. In CHO cells, however, there was no increase in thioguanine-resistant cells at dose levels that reduced cell survival to < 20%. These results are consistent with published reports on PAP. While the CHO cells were slightly more resistant to the toxic effects of PAP, the dose levels used in the two cell lines did not differ by more than 2-fold. At equivalent survival levels, PAP induced a significant (up to 20% aberrant cells) number of aberrations, primarily complex rearrangements, in both cell lines. In the single cell electrophoresis assay, there was a reproducible dose-related increase in cells with single-strand DNA breaks with both the L5178Y cells and the CHO cells. The induction of single-strand breaks and chromosome aberrations by PAP suggests that, mechanistically, PAP produces similar genetic damage in both CHO and L5178Y cell lines, but intrinsic differences between assay systems are responsible for the divergent gene mutation results.

Effects of plate preparation on results in microbial mutation assays.

Glucose autoclaved in an alkaline phosphate solution (heated glucose+salts, HGS) results in the production of a moiety that is nonmutagenic but can interact with a series of 4-[2-(aryl)ethenyl]-2,6-dimethylphenols to result in an increase in bacterial revertants that is dependent on the amount of HGS in the minimal agar plates. The reaction between the HGS and the chemical to form a mutagen is independent of the presence of bacteria, does not result in a nutritive analog to enhance growth of the auxotrophic bacteria, and is effective only in Salmonella typhimurium and Escherichia coli strains that contain the plasmid pKM101. A sufficient amount of this glucose product may be formed in normal plate preparation to produce apparent mutagenic activity of these chemicals.

Development of an optimal S9 activation mixture for the L5178Y TK+/- mouse lymphoma mutation assay.

In previously described activation systems [Clive D, Spector JFS (1975): Mutat Res 31:17-29] for the mouse lymphoma mutation assay the cofactor isocitrate is rapidly exhausted and the resultant loss of NADPH can halt metabolic processes. Presented here are data obtained with a non-toxic balance of NADP (1.4 mg/ml), isocitrate (6.0 mg/ml), and S9 (less than or equal to 4%) in Fischer's medium which produces a more stable supply of the required cofactors. By spectrophotometric analysis, the molar concentration of NADPH remains at greater than or equal to 50% or more of the maximum over the usual 4-hr treatment period. Accompanying this increase in NADPH duration was increased toxicity and mutant frequency at most doses among cells treated with the reference mutagens 3-methylcholanthrene (MCA), 2-acetylaminofluorene (AAF), benzo(a)pyrene (BAP), 9,10-dimethyl-1,2-benzanthracene (DMBA), or cyclophosphamide (CPA), but not with dimethylnitrosamine (DMN)-possibly a reflection of the single enzyme mediated step in the metabolism of this chemical. These observations also suggest that results attributed to varying the amounts of S9 in an activation mixture may be due to suboptimal cofactor levels and further emphasize the need to maintain sufficient NADPH exposure to evaluate the effects of metabolic enzyme levels or compare the relative activities of analogous chemicals.